Effects of prolactin on conceptus survival and uterine secretory activity in pigs

Hypoprolactinaemia was induced by bromocriptine (CB154; 100 mg/day) which decreased circulating prolactin by 40% (P less than 0.06), but did not affect conceptus survival at Day 25 when administered on Days 10-16 when compared to saline:ethanol-treated control gilts. Bromocriptine or vehicle was administered to cyclic gilts on Days 10-11, oestradiol valerate was injected on Day 11 and uterine flushings were collected on Day 12. Total recoverable protein and uteroferrin in uterine flushings were not affected by treatment. However, leucine aminopeptidase activity (P less than 0.02) and total recoverable Ca2+, Na+, K+ and Cl- (P less than 0.05) were decreased in uterine flushings of gilts that received bromocriptine, suggesting that hypoprolactinaemia decreased general secretory activity of the endometrial epithelium and modulated ionic changes, respectively, in the uterine environment of pigs. Subcutaneous administration of pig prolactin (1 mg/12 h) increased (P less than 0.001) serum prolactin 4.5-fold. The interaction between hyperprolactinaemia and progesterone, without oestrogen, on components of uterine flushings were determined using gilts that received progesterone (200 mg/day) and prolactin or saline on Days 4-14 after ovariectomy on Day 4. On Day 15, there were no differences (P greater than 0.05) in any of the uterine secretory components measured. Hyperprolactinaemia (1 mg pig prolactin on Days 6-11) enhanced overall uterine secretory response on Day 12 to oestradiol (5 mg) administered on Day 11 compared to gilts that received 1 ml saline on Days 6-11 of the oestrous cycle. Total recoverable protein and leucine aminopeptidase activity were greater (P less than 0.05) for oestradiol-treated gilts, but effects of prolactin were not significant. Total recoverable glucose (P less than 0.01), PGF-2 alpha (P less than 0.02), uteroferrin (P less than 0.01) and specific activity of uteroferrin (P less than 0.001) were increased by prolactin and oestradiol, but not oestradiol alone. Calcium (P less than 0.05), chloride (P less than 0.05) and potassium (P less than 0.01) were increased in response to oestradiol. These results indicate an interaction between oestradiol and prolactin, but not progesterone and prolactin, which enhances secretion of some products of the pig uterine endometrium.     

Steroid metabolism by endometrial and conceptus tissues during early pregnancy and pseudopregnancy in gilts

Endometrial and conceptus tissues were obtained on Days 10.5, 11, 12, 16 and 25 of pregnancy and Day 25 of pseudopregnancy of gilts and incubated for 6 h in Minimal Essential Medium (5 ml) containing 35 ng [3H]progesterone. Metabolism of [3H]progesterone to oestrone, oestradiol and oestriol was determined by gas and high-pressure liquid chromatography and successive recrystallizations with unlabelled standards. Conceptuses collected between Days 10.5 and 12 were spherical, tubular or filamentous and incubated with 500 mg endometrium and [3H]progesterone. Production of oestrone by spherical conceptuses was not detected, but was 44-47 pg/tubular conceptus and 21 pg/filamentous conceptus. A similar trend was observed for oestradiol. Conceptus tissues from Days 16 and 25 (chorion) were most active in producing oestrone (123 and 520 pg/mg tissue, respectively) and oestradiol (277 and 876 pg/mg tissue, respectively). Endometrial oestrogen production was less than that for conceptus tissue for oestrone and oestradiol on Days 16 and 25 of gestation. Coincubations of endometrium and conceptus tissues had lower oestrogen production than conceptus alone. Endometrium from Day 25 of pseudopregnancy metabolized [3H]progesterone to several non-polar metabolites, but no oestrogens were detected. An unidentified phenolic metabolite of [3H]progesterone was detected in higher quantities than either oestrone or oestradiol; 445 to 461 pg/conceptus at the tubular stage. These results indicate temporal changes in the conversion of [3H]progesterone to oestrogens by conceptus and endometrial tissue from pregnant gilts, but not endometrium from pseudopregnant gilts.     

Evidence for multiple uterine modulators of rabbit luteal function: the effect of hysterectomy during pseudopregnancy in the estrogen-treated rabbit

Previous studies show that hysterectomy on Day 1 of pseudopregnancy prolongs serum progesterone secretion in estrogen-treated pseudopregnant rabbits. These studies were undertaken to determine the day of pseudopregnancy when uterine factors are released to alter luteal function. When hysterectomies were performed on either Day 5, 8, 10, or 13 of pseudopregnancy, serum progesterone concentrations were greater than 10 ng/ml between Days 18 and 27 of pseudopregnancy compared to levels of approximately 4 ng/ml in sham-hysterectomized rabbits on these same days. In contrast, serum progesterone levels were not elevated when hysterectomies were performed on Day 11 of pseudopregnancy and were only partially maintained when hysterectomies were performed on Day 12 of pseudopregnancy. Twice daily injections of prolactin (1.5 mg, s.c.) between Days 1 and 33 of pseudopregnancy were unable to mimic the effect of estradiol in the hysterectomized rabbit. Twice daily injections of indomethacin (8 mg/kg, s.c.) between Days 6 and 23 of pseudopregnancy lowered uterine and luteal prostaglandin F2 alpha levels approximately 10-fold on Day 24 of pseudopregnancy but did not maintain progesterone secretion. Serum cholesterol levels were not altered by hysterectomy on any day and were thus not related to the maintenance of progesterone production. These results suggest that the uterus produces both inhibitory and stimulatory factors that effect luteal progesterone secretion. First, an inhibitor is released between Days 10 and 11 of pseudopregnancy in estrogen-treated rabbits that prevents the rabbit corpus luteum from responding to estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oestradiol administration raises luteal LH receptor levels in intact and hysterectomized pigs

Occupied and unoccupied LH receptors in corpora lutea, and LH and progesterone concentrations in circulating plasma, were measured in non-pregnant gilts that had been treated with oestradiol-17 beta benzoate to prolong luteal function. Oestradiol benzoate (5 mg, administered on Day 12 after oestrus) delayed luteal regression and the decline in LH receptor levels at luteolysis and raised unoccupied receptor levels from 11.8 +/- 1.14 fmol/mg protein on Days 10--15 after oestrus to 31.8 +/- 3.26 fmol/mg protein on Days 15--21. There was no simultaneous rise in occupied receptor levels and occupancy decreased from 29.8 +/- 3.01 to 11.5 +/- 1.26%. Basal plasma LH concentrations were unchanged by oestradiol, but mean corpus luteum weight and plasma progesterone concentrations were slightly reduced. Oestradiol benzoate on Day 12 caused a similar increase in unoccupied receptor levels in gilts hysterectomized on Days 6--9 after oestrus, from 17.0 +/- 5.83 to 34.5 +/- 6.00 fmol/mg protein, determined on Days 15--18. Plasma concentrations of LH and progesterone were unchanged by oestradiol. Unoccupied receptor levels in corpora lutea and plasma LH and progesterone were unaltered by hysterectomy in untreated gilts. Occupied receptor levels were not influenced by hysterectomy or oestradiol. It is concluded that oestradiol-17 beta raises luteal LH receptor levels by a mechanism independent of the uterus.     

Development and survival of pig blastocysts after oestrogen administration on day 9 or days 9 and 10 of pregnancy

In Exp. 1, administration of 5 mg oestradiol valerate i.m. to pregnant gilts on Days 9 or 9 and 10 advanced the uterine secretion of calcium, protein, and acid phosphatase as demonstrated by levels recovered in the uterine flushings of females unilaterally hysterectomized on Day 11. Upon removal of the remaining uterine horn on Day 12, protein and acid phosphatase increased while Ca2+ decreased in oestradiol-treated gilts as did PGF. In contrast, a 4-fold increase in recoverable Ca2+ occurred from Days 11 to 12 in control gilts. Recoverable oestradiol-17 beta was increased in all 3 groups on Day 12 and plasmin inhibitor concentration increased in oestradiol-treated gilts. Two-dimensional PAGE demonstrated the appearance of a group of very acidic polypeptides in oestradiol-treated gilts. Blastocysts recovered from the second uterine horn had undergone elongation to the filamentous morphology in all 3 groups. In Exp. 2, oestradiol valerate was administered to pregnant gilts on Day 9 or Days 9 and 10 followed by total hysterectomy on Day 16. No differences in recoverable Ca2+ or protein were found, but acid phosphatase was decreased by 75% after oestradiol treatment. Recoverable oestradiol was decreased in oestradiol-treated gilts while PGF and plasmin inhibitor concentrations were unaffected. Compared with the control gilts, blastocysts recovered from oestradiol-treated gilts were fragmented and degenerating on Day 16. PAGE demonstrated greatly intensified staining of the group of acidic polypeptides in oestradiol-treated gilts. These results indicate that oestradiol treatment on Day 9 of pregnancy advances uterine secretory response, but that blastocyst elongation can occur in this uterine environment and in the presence of declining intraluminal Ca2+ levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pregnancy, injection of oestradiol benzoate or hCG on steroid concentration and release by pig luteal cells

Corpora lutea were obtained from pig ovaries on Day 18 of pregnancy or pseudopregnancy. Pseudopregnancy was induced by the administration of oestradiol benzoate on Days 11-15 of the oestrous cycle or by the administration of hCG on Day 12. The luteal cells were prepared for morphometric analysis and investigation of steroid production in vitro by dispersion with 0.25% trypsin. A blood sample from each sow was collected at slaughter for measurement of progesterone, oestradiol-17 beta and testosterone. The concentrations of these steroids were also estimated in luteal tissue and in the medium after incubation. Progesterone concentration was significantly higher (P less than 0.01) in luteal tissue and in plasma of pregnant than of pseudopregnant sows. Testosterone content of luteal tissue from all sows was 20-fold higher than oestradiol, although plasma concentrations of these hormones were not different. The luteal cells from hCG-treated sows produced more progesterone (P less than 0.01) in vitro than did those from the other groups. The luteal cells from oestradiol-treated sows generally released smaller amounts of steroids during incubation. Treatment with hCG increased the proportion of large luteal cells and decreased the proportion of small luteal cells. These results demonstrate that hCG or oestradiol benzoate injections altered the steroidogenic activity of luteal cells and that treatment with hCG was also associated with changes in the diameter of the luteal cells and thus in the ratio of small to large luteal cells.     

Critical progesterone requirement for maintenance of pregnancy in ovariectomized rats

The minimum progesterone concentration required to maintain the pregnancy was studied by varying doses of progesterone given subcutaneously to rats ovariectomized on Day 8 of pregnancy. Injecting 3 mg progesterone plus 200 ng oestradiol benzoate daily provided serum progesterone values between 25.4 +/- 7.0 and 35.2 +/- 6.2 ng/ml throughout Days 10-19 which were significantly lower than normal levels (P less than 0.05), but resulted in 93.6% of fetal survival on Day 19 which was not significantly different from 93.3% in the control group. Injecting 2 mg progesterone plus 200 ng oestradiol benzoate daily gave progesterone values between 13.2 +/- 4.6 and 19.0 +/- 6.2 ng/ml and could not maintain fetal viability to Day 19 (14.2%, P less than 0.05 compared with control group). Critical times to supplement progesterone in rats ovariectomized on Day 8 or Day 15 were studied by varying the time of progesterone implantation after ovariectomy. Progesterone implants were administered 8, 12 and 24 h after ovariectomy on Day 8 and 24, 36 and 48 h after ovariectomy on Day 15. On Day 8, progesterone replacement could be delayed to 8 h but not 12 h, while on Day 15, progesterone replacement could be delayed up to 36 h but not 48 h after ovariectomy without affecting fetal survival.     

Quantitative light microscopic analysis of corpus luteum growth during pseudopregnancy in the rabbit

We employed stereological methods at the light-microscope level to examine the mechanism by which corpora lutea (CL) grow during the course of pseudopregnancy in the rabbit. Corpus luteum volume per ovary, the absolute volume of luteal cells per CL, individual luteal cell volume, the number of luteal and endothelial cells per CL, and capillary surface area per CL were examined in rabbits at Days 1, 4, 7, 11, and 18 of pseudopregnancy. Total CL volume increased from 3.7 +/- 0.1 microliter to 30.3 +/- 0.5 microliter over Days 1 to 11 and thereafter decreased to 15.2 +/- 1.1 microliter by Day 18. Stereological analyses showed that the increases in CL volume from Day 1 to Day 11 were due primarily to increases in the volume of individual luteal cells (from 2.6 +/- 0.2 pl on Day 1 to 23.5 +/- 1.7 pl on Day 11, 1 pl = (10 mu)3; r = 0.96), and that the decrease in CL volume after Day 11 resulted largely from a decrease in luteal cell volume (to 12.8 +/- 1.5 pl). In contrast, no change was seen in the number of luteal cells per CL (range 9.1 x 10(5)-12.5 x 10(5)). These data show that CL growth and subsequent regression during pseudopregnancy result primarily from changes in the volume of individual luteal cells, and not from changes in the number of luteal cells. These data support the hypothesis that modulation of progesterone production during pseudopregnancy is due to changes in individual luteal cell volume and not to changes in cell number.     

Change in responsiveness of rabbit corpus luteum to prostaglandin F-2 alpha during pregnancy and pseudopregnancy.

Previous studies have suggested that prostaglandin F-2 alpha (PGF-2 alpha) may have a role in luteolysis in rabbits. Rabbits (4-6/group) were given a single injection of saline, or 100, 500 or 2500 micrograms PGF-2 alpha (i.m.) on Day 7, 9, 12 or 15 of pregnancy or pseudopregnancy. Daily blood samples were taken via the marginal ear vein before and for 3 days after the PGF-2 alpha injection. Concentrations of serum progesterone were determined by radioimmunoassay in pseudopregnant rabbits. There were no significant differences between PGF-2 alpha-treated and control rabbits on Days 7 or 9. On Day 12 of pseudopregnancy, progesterone concentration was significantly (P less than 0.05) lower in treated than in control rabbits, the effect being dose dependent. On Day 15 of pseudopregnancy, it was not possible to distinguish between controls and treated groups because luteolysis occurred in all rabbits. In contrast, on Days 7 and 9 of pregnancy, the concentration of progesterone in treated groups was lower than in the control groups (P less than 0.05), the effect being dose dependent. This difference was maintained throughout the sampling period and resulted in termination of pregnancy. By Day 12 of pregnancy, the response to PGF-2 alpha was transient, with a significant decline in progesterone for only 2 days, followed by a return to control concentrations and normal delivery of litters. On Day 15 of pregnancy, no treatment with PGF-2 alpha significantly altered progesterone concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative roles of oestradiol and of the uterus in the maintenance of the corpus luteum in the pseudopregnant brown hare (Lepus europaeus)

Brown hares were made pseudopregnant by sterile matings or PMSG-hCG treatment (day of mating or hCG injection = Day 0 of pseudopregnancy). Progesterone secretion by the CL began 3-4 days after the ovulatory stimuli, reached maximum on Days 8 to 11 and decreased thereafter to reach low levels from Day 9 to 18, depending on the female. Cauterization of all large ovarian follicles on Day 7 resulted in an immediate luteolysis in young females, but had no effect in older ones. Oestradiol capsules implanted from Day 7 to Day 46 were able to maintain progesterone secretion until at least Day 30, in intact females as well as in females with all large follicles cauterized. Hysterectomy on Day 7 or 8 was followed by an immediate drop in progesterone concentrations; oestradiol capsules implanted at the time of hysterectomy prevented the drop in progesterone values, which remained elevated until Day 38. The induction of ovulation in females hysterectomized 2 months before resulted in CL of slightly shortened life-span. The injection of PGF-2 alpha on Day 7 of pseudopregnancy was followed by an immediate luteolysis. These results suggest that oestradiol secreted by the large ovarian follicles is the main luteotrophic factor in the brown hare. In old hares, the large amount of interstitial tissue could secrete oestrogens, and thus maintain pseudopregnancy. On Day 7 of pseudopregnancy, the uterus secretes a luteotrophic substance acting either directly on the ovary, or via the pituitary, to maintain oestradiol secretion by the follicles. In long-term hysterectomized females, the CL would be able to develop independently of any trophic substance, but for a reduced duration.     

Effects of oestrogen supplementation and space restriction on PGF2alpha-induced nest-building in pseudopregnant gilts.

This study examined the role of oestrogen supplementation on PGF2alpha-induced nest-building in pseudopregnant gilts. Oestradiol valerate (5 mg/day) injections were given on Days 11-15 of the oestrous cycle to induce pseudopregnancy. A further series of injections of either oestradiol valerate (5 mg/day) or vehicle were given on days 44-46 of pseudopregnancy to reflect more closely the hormone profile seen in pregnancy. Nest-building was induced by a single intramuscular injection of 15 mg of PGF2alpha (Lutalyse) on Day 47 of pseudopregnancy. The gilts were housed in pens (2.8 x 1.7 m) containing straw in experiment 1 or chronically confined in crates (0.6 x 1.7 m) that did not contain straw on days 44-48 of pseudopregnancy for experiment 2. Oestrogen supplemented gilts had significantly higher concentrations of circulating 17beta-oestradiol on day 47 of pseudopregnancy but there were no significant differences between treatments for circulating levels of prolactin, progesterone, cortisol or oxytocin, or for any behavioural measure in either experiment. These results indicate that there is no direct effect of supplementing already pseudopregnant gilts with oestradiol valerate on PGF2alpha-induced nest-building. The results also show that the pre-partum environment has a pronounced effect on nest-building behaviours and that non-pregnant pigs might be a useful model for pre-partum nest-building in this species.     

Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of oestrogen induced morphological changes in the rabbit corpus luteum

Although oestradiol is luteotropic in the rabbit, the mechanism through which oestradiol maintains the corpus luteum is unknown. To determine if ultrastructural changes in the rabbit corpus luteum accompany oestradiol treatment, corpora lutea were removed on day 10 of pseudopregnancy from hysterectomized rabbits treated with oestradiol implants and prepared for electron microscopy. Morphometric analysis was performed on luteal cells in each group (seven animals/group, four micrographs/animal) and organelle profile volumes, estimated by point-counting techniques, were expressed as a percentage of the cell volume. Morphometric analysis revealed no significant differences in nuclear, mitochondrial, lysosomal, Golgi or endoplasmic reticular volumes between control and oestradiol-treated animals. In contrast, lipid droplet volume was significantly elevated (p less than 0.001) in oestradiol-treated animals. This data suggests that an important function of oestradiol in the rabbit corpus luteum is to regulate the uptake and storage of cholesterol presumably for progesterone production.     

Effect of mifepristone (RU 486) on concentrations of prostaglandin E-2 binding sites in the rat endometrium

Progesterone implants in ovariectomized rats increased endometrial concentrations of PGE-2 receptors. The increase was completely inhibited by simultaneous daily injection (7.5 mg/kg) of mifepristone (RU 486). A single injection of mifepristone on the morning of Day 1 of pseudopregnancy (day of oestrus) decreased the amount of PGE-2 receptors found in the endometrium on Day 5 by 64%. This inhibitory effect probably resulted from the antiprogesterone activity of this compound since it was not counteracted by simultaneous treatment with dexamethasone, shown to reverse totally the antiglucocorticoid action of mifepristone. The inhibition by mifepristone lasted only for 1 day; endometrial PGE-2 receptor levels on Day 6 of pseudopregnancy returned to the high values present in controls. Under these conditions, administration of the mifepristone did not affect the plasma oestradiol and progesterone concentrations during the 1st week of pseudopregnancy. The administration of mifepristone on Days 2 and 3 of pseudopregnancy kept the endometrial PGE-2 receptor levels low, even by 4 days after the end of treatment. We therefore concluded that, in the rat, progesterone priming leading to uterine receptivity can be delayed, at least by 1 day. In contrast, interruption of the progesterone action for a longer period later during the early pseudopregnant period resulted in an altered subsequent evolution of the endometrium, in terms of acquisition of the PGE-2 binding sites.     

Adenylate cyclase activity of the corpus luteum during the oestrous cycle of the pig

Basal adenylate cyclase values for corpora lutea (CL) removed from cyclic gilts on Days 3, 8, 13 and 18 were 178 +/- 61, 450 +/- 46, 220 +/- 25 and 208 +/- 18 pmol cAMP formed/min/mg protein, respectively. Basal activity was significantly elevated on Day 8 (P less than 0.001). LH-stimulatable adenylate cyclase values for CL from Days 3, 8, 13 and 18 were 242 +/- 83, 598 +/- 84, 261 +/- 27 and 205 +/- 17 pmol cAMP formed/min/mg protein respectively. Serum progesterone concentrations of 12 gilts bled every 2 days through one complete oestrous cycle ranged from 1.1 to 26.9 ng/ml with highest values between Days 8 and 12. The decline in serum progesterone concentrations was coincident with the decrease in basal adenylate cyclase activity. There was no LH-stimulatable adenylate cyclase activity present in the CL at the specific times of the oestrous cycle examined. We conclude that progesterone secretion by the pig CL is apparently dependent on basal activity of adenylate cyclase.     

Isoprenoid synthesis in Halobacterium halobium. Modulation of 3-hydroxy-3-methylglutaryl coenzyme a concentration in response to mevalonate availability

Halobacterium halobium was evaluated as a potentially simpler biological model to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (content) in response to mevalonate availability. H. halobium's HMG-CoA reductase was soluble and required NADPH as its reduced coenzyme. Maximum HMG-CoA reductase activity (4-10 nmol/min/mg of soluble protein) was obtained in buffers which contained 3.5 M KCl. Mevinolin (a) blocked growth of H. halobium, (b) was a competitive inhibitor of HMG-CoA reductase (Ki = 20 nM), (c) did not cause the paradoxical increase in assayable reductase activity, as reported for eukaryotic cells, and (d) caused a rapid (within 30 min) 8-12-fold accumulation of intracellular HMG-CoA. Mevalonate blocked and reversed mevinolin-mediated HMG-CoA accumulation. Although mevinolin-treated cell's growth was restored by mevalonate, HMG-CoA reductase's activity was not. Thus, H. halobium is a unique biological model which allows one to study the regulation of intracellular HMG-CoA concentration and not HMG-CoA reductase activity (content) in response to mevalonate availability.     

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation.

Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8.     

Effects of corpus luteum removal on progesterone, estradiol-17 beta and LH in early pregnancy of the tammar wallaby, Macropus eugenii

The quiescent corpus luteum of female tammars was reactivated by removal of the pouch young (RPY). The reactivated corpus luteum was ablated 3 days after RPY. Plasma progesterone and oestradiol concentrations were measured by radioimmunoassay in these and in sham-operated controls. Excision of the CL abolished the rise in progesterone seen at Day 5-6 in the sham-operated animals (130.7 +/- 56.6 vs 452.4 +/- 176.0 pg/ml, mean +/- s.d.). By contrast, oestradiol-17 beta values increased within 6-16 h of CL excision to 16.3 +/- 6.9 pg/ml and remained high for 1-3 days while in the sham-operated animals there were less sustained and more variable peaks of 10-20 pg/ml between Days 3 and 5 (mean 12.0 +/- 3.6 pg/ml at Day 4-5). We conclude that the early transient increase in peripheral plasma of progesterone is of luteal origin but the source of the oestradiol remains unknown.     

Effect of RU 486 on luteal function in the early pregnant rat

A dose of 30 mg RU 486/kg, an antiprogesterone, was administered to pregnant rats on Day 2 (Group 1) or Day 4 (Group 2) of pregnancy. RU 486 significantly changed serum progesterone and oestradiol concentrations and luteal 3 beta-HSD and 20 alpha-HSD activities in Group 1, and implantation was significantly inhibited. The luteal 3 beta-HSD activity in Group 2 rats on Day 6 was significantly (P less than 0.01) lower than the control value (7.5 +/- 0.6 and 10.1 +/- 0.6 mU/mg protein respectively). This decline in the 3 beta-HSD activity was followed by a marked decrease in the serum progesterone concentration, resulting in a significant decrease of the progesterone/oestradiol ratio and implantation was completely inhibited. The 20 alpha-HSD activity, which could not be detected on Day 6 in the control rats, was twice as great in Group 2 than in Group 1 rats (17.5 +/- 1.2 and 7.4 +/- 3.1 mU/mg protein respectively). Ultrastructural examination of corpora lutea of Group 2 rats confirmed luteolysis. These results suggest that RU 486 has a luteolytic effect and its anti-implantation effect is concomitant with luteolysis of the corpora lutea of pregnancy.